Microfluidics based assay device

ABSTRACT

A subtractive corrective assay device and methodology, whereby ail required binding and label detection reagents are initially located within the detection zone. Application of a magnetic field is used to selectively remove bound label from the detection zone by means of paramagnetic particles. The relationship between measured label concentration before and after the application of a magnetic field within the detection zone is used to accurately measure analyte concentration within the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage entry under 35 U.S.C. § 371(b) ofInternational Application No. PCT/GB2012/000122, filed Feb. 7, 2012,which claims the benefit of United Kingdom Patent Application Serial No.1102037.7, filed Feb. 7, 2011, the disclosures of both of which arehereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a microfluidic based assay system,comprising a disposable assay cartridge and associated reading device,as well as the individual components themselves. The present inventionalso relates to methods of conducting assays, using the cartridge anddevice of the invention, as well as kits for conducting assays.

BACKGROUND TO THE INVENTION

There is a constant need within the IVD industry to deliver improvementsin performance. Improvements in performance can include accuracy,precision, cost, multiplexing, total test time etc. The novelsubtractive corrective assay device and method described in thisdocument is designed to deliver such improvements. The novel subtractivecorrective assay device and method is composed of a single usedisposable strip and a reusable reader. Although other detectionmethodologies are applicable, the preferred embodiment focuses onelectrochemical detection.

In the prior art there are numerous examples whereby magnetic particlesand associated bound species which give detectable changes are broughtto a detection zone via a magnetic field. Typically the unbound label iskept away from the detection zone with the bound label being accumulatedwithin the detection zone and inducing the detectable change.

US 2009/0130771 A1 incorporated by reference in its entirety, describesthe use of magnetic particles to capture analyte but accumulates themwithin a detection zone to measure an increased concentration of analytewithin the detection zone. US 2009/0130771 A1 also uses a separatereference zone for any background correction. This only works forcorrection of background of the sample itself and not for any variationsin label or other reagent concentrations as the two areas are differentand therefore differences will be present with respect to resuspendedreagent concentrations as well as the reproducibility of measuringwithin 2 distinct areas, such as reference vs detection zone size,opacity etc. US 200910130771 A1 does make reference to the possibilityof using the detection zone for a background measurement but only when“GOD [label] particles are substantially absent from the detection zone”and therefore does not correct for variation in label concentration.

It is amongst the objects of the present invention to provide a cheapand reliable assay system for carrying out IVD tests.

It is amongst the objects of the present invention to provide an assaycartridge design platform and reader which may be easily and cheaplyfabricated, as well as being able to be configured to carry out aspecified assay or assays.

It is amongst the objects of the present invention to provide an assaycartridge which may easily be adapted to carry out a variety ofdifferent specified assays.

It is amongst the objects of the present invention to provide an assaysystem comprising a reader which may preferably be used or easilyadapted to perform a variety of different assays.

SUMMARY OF THE INVENTION

The present invention is based on the development of a novel subtractiveassay device and method whereby all the reagents are initially locatedwithin the detection zone. The assay architecture allows for veryaccurate, sensitive measurements whereby the concentration of theunbound label is used to measure the concentration of the analyte(s).

In a first aspect the present invention provides a microfluidic assaycartridge for use in a subtraction assay for detecting an analyte in asample of fluid, the cartridge comprising:

-   a sample port for introducing said fluid sample into the cartridge,-   a substrate comprising one or more microfluidic channels disposed    therein and comprising a binding agent disposed within said    channel(s) for binding any of said analyte within the sample and a    label for use in detecting an amount of the analyte present in the    sample; and-   a detection area within or wholly comprising an area where analyte    and label binding occurs, said detection area from which bound    analyte is removed allowing determination of said analyte    concentration indirectly by measurement of any remaining unbound    label.

The detection zone contains the reagents required for the bindingreaction and an enzymatic reaction to occur simultaneously uponrehydration of the reagents by addition of the sample. In a specificembodiment, specifically anti-analyte magnetic particles, anti-analyteglucose oxidase (GOD) label and mediator system are located within thedetection zone. Glucose is deposited upstream from the detection zone soupon cartridge filling by the sample, the glucose is rehydrated andpresented with sample to the detection zone(s). The sample (withglucose) rehydrates the binding and enzymatic reagents. Immediately bothreactions start (formation of bound complexes and enzymatic turnover ofsubstrate). A reference measurement of enzyme turnover is then performedwhich takes into account a number of variables in the assay(anti-analyte-GOD label concentration, mediator concentrations, workingelectrode size etc). This reference measurement is dominated by theanti-analyte-GOD label concentration. Hereinafter, this is termed thepre magnetic separation measurement.

After a defined period of time (e.g. 4 minutes) a magnetic field isapplied to the strip. As a result the paramagnetic particle-analyte andparamagnetic particle-analyte-label complexes are selectively removedfrom the detection zone to the source of the magnetic field leavingunbound GOD label in the detection zone. After a defined period of time(e.g. two minutes) a measurement is performed measuring theconcentration of the unbound GOD label remaining within the detectionzone. The relationship between the label concentration before and afterthe magnetic separation step is used to determine the concentration ofanalyte(s) within a sample.

There are a number of advantages to performing a subtraction correctiveassay which measures the label concentration before and after themagnetic separation. A great source of variation and inaccuracy withindisposable IVD strips is the amount/concentration of reagents depositedin the cartridge. The corrective subtractive assay methodology correctsfor this because each of the reagents is deposited within the detectionzone allowing determination of label concentration and hence the abilityto correct for differences in label concentrations. This worksexceptionally well for a subtraction method as only events at a workingelectrode are being interrogated (i.e. the label concentration at theworking electrode is measured and then after the magnetic removal of thebound label, the unbound label concentration at the working electrode isre-measured). In accumulation assays where detectable species are boughtto the electrode there is no opportunity for correction, even if each ofthe reagents were located in the detection area; bound species wouldstill be brought to the working electrode (and hence the inability tocorrect for variations in label concentration elsewhere in the strip).This does not occur in the corrective subtraction assays of the presentinvention and therefore it allows very accurate corrections forvariations in label concentrations (and other sources of variationpreviously described) resulting in improved precision, sensitivity,accuracy and overall assay performance (the corrective ability isprofound on the assay performance as further described in the detaileddescription).

An additional major advantage of the subtractive corrective methodologyis that of independence of strip volume. In assay methodologies wherebylabel is brought to the detection zone via magnetic particles themeasurement is susceptible to volume changes of the strip (width, heightetc) of the channel (i.e. the amount of analyte available for bindingvaries with the variation of the dimensions of the strip). In the caseof the subtractive corrective methodology the volume of the strip isnormalised. This is driven by the pre magnetic separation measurementwhereby only the species at the working electrode surface is measured.This measurement therefore allows for correction in strip volume as onlythe sample volume at the working electrodes is measured, thereforevariations in strip channel height, strip width, strip volume, electrodesize and reagent concentrations can be corrected for resulting in highlyaccurate results.

Assays using accumulation of paramagnetic particles to bring bound labelto a detection area also suffer from difficulties in collecting all (ora reproducible number) of the paramagnetic particles and presenting themto the detector in a reproducible manner (for example paramagneticparticle bead band dimensions) to ensure accurate results. Thesubtraction methodology does not suffer from this as the paramagneticparticles and bound species are removed from the detection area.

As the assay methodology is very simple it is easy to create cartridgeformats capable of multiple measurements. This allows measurements ofmany different analytes or multiple measurements of the same analytes.In addition the simplicity of the assay allows a highly manufacturablecheap cartridge design that uses very small sample volumes and istherefore applicable to many product applications

The cartridge design of the present invention may easily be adapted tocarry out a number of different assays and hence can be considered as anassay platform for a variety of assays. The cartridge and channel(s)disposed therein may be formed in any manner of ways known to theskilled addressees, which may include photolithography, wet chemicaletching, laser ablation, injection moulding, embossing and printingtechniques. However, in a preferred embodiment, the cartridge and thechannels and other features disposed therein, are formed by a sandwichof three separate substrates—a top, middle and bottom substrate.

The cartridge can be formed of any suitable material, such aspolycarbonate, polyester, polystyrene, PMMA, etc. and the/each substratemay be formed of a single or plurality of material(s). In the embodimentcomprising three substrates, the middle substrate comprises a patterncut through the substrate, corresponding to certain features of thecartridge, such as the channel(s), sample introduction port and thelike. By applying and sandwiching (such as by heat sealing, gluing,stapling and the like) appropriately cut top and bottom substrates, tosandwich the middle substrate between the top and bottom substrates, acartridge can be provided in which channels and other features aredisposed. Openings or features in the top and/or bottom substrate may bedesigned to allow air to vent from the cartridge to allow filing withsample or co-locate with features in a reader device (as will bediscussed hereinafter), which may facilitate with correct location ofthe cartridge in the reader.

As identified, in use, the sample is applied to the cartridge through asample introduction port such as by way of capillary action. In apreferred embodiment the sample introduction port is an aperture in aside or face of the cartridge. Desirably the cartridge is in the form ofa generally thin planar device comprising top and bottom faces and fouredges. In this arrangement, the sample introduction port may be formedin one of the edges of the cartridge, so that a user need only contactthe sample with the aperture formed in the edge, in order to enablesample uptake into the cartridge. In use the user contacts the fluidsample with the port/aperture and, in certain embodiments, due to thedimensions of said channel(s) within the cartridge, fluid is drawn intothe cartridge by capillary action. The dimensions of the sampleport/aperture may be smaller or larger than the dimensions of thechannel(s).

Said channel(s) in the cartridge may also comprise one or more fluidstop features, which are designed to prevent the sample and/or otherfluids from passing through the stop feature, by virtue of capillaryaction alone. A preferred stop feature is a hydrophobic material (e.g.printable conductive or non conductive inks) or a process or materialthat changes the surface properties of a channel surface thereforecreating a hydrophilic/hydrophobic differential (e.g. by way of laserablation, surface scoring, surface material removal, evaporated metallicmaterials etc), which is designed to abut/be a wall feature or is coatedon a wall of the channel or an air gap in the channel (for example ahole in the lid and/or base material which spans the channel). In theembodiment where the channels are formed by virtue of three substratesbeing sandwiched together thereby forming the channels, the hydrophobicmaterial may be applied to the top and/or bottom substrates, such thatwhen the three substrates are sandwiched together, the hydrophobic stopmaterial forms a feature on the top and/or bottom surface of saidchannel.

As well as the microfluidic channel(s), the cartridge of the presentinvention may comprise one or more electrode features which contact withthe channel and hence the sample once introduced into the cartridge. Theelectrodes are designed to contact electrical contacts within thereader, enabling a variety of readings to be taken, where appropriate.For example, one or more electrodes in the cartridge may be designed todetect correct loading of the cartridge and the reader may signal to theuser whether or not the cartridge has a) been correctly inserted intothe reader and/or the sample loaded into the cartridge correctly. Theelectrode(s) may also carry out one or more electrical measurements onthe sample itself. For example, when the sample is a sample of wholeblood, the electrode(s) may conduct a hematocrit measurement of thesample, which may be important in determining an accurate concentrationof the analyte to be detected. Conductivity and/or impedancemeasurements may be determined depending on the sample being studied.The label used in analyte detection may be electrochemical (or involvedin an electrochemical reaction) and therefore these electrodes may beused in the measurement of the label concentration and therefore analyteconcentration. Thus, the cartridges of the present invention may useelectrical measurements on the sample for functions such as filldetection, hematocrit measurement and analyte measurement.

Any conductive material could be used to form electrodes. For examplescreen printable carbon inks, silver/silver chloride inks, gold,platinum, copper etc could be used and applied to a substrate by variousmeans such as screen printing, sputtering, ink jet printing etc orpartial removal of electrode material from a substrate by various meansincluding chemical etching, laser ablation etc.

The sample to be applied to the cartridge may be any suitable fluidsample. It may for example be a sample of fluid obtained from a subject,such as a whole blood, plasma, saliva, semen, sweat, serum, menses,amniotic fluid, tears, a tissue swab, urine, cerebrospinal fluid, mucousand the like. It is to be appreciated that the assay systems of thepresent invention may be applied in the human health area, includinglarge and growing IVD markets (e.g. cancer, cardiology, diabetes andinfectious disease). The assays may also be used to test drugs and drugaction. However, the system may also be applied in environmentalsettings where it is desirable to detect, for example toxic agents orinfectious agents such as bacteria or viruses. Thus, samples from riversor lakes or swabs from solid surfaces may be taken in order to obtain afluid sample for providing to the cartridge. The assay systems may alsobe utilised for veterinary applications for laboratory, point of careand in the field testing. Essentially any assay in which a sample can beprovided in a fluid form may be utilised in the present invention.

The sample may, for instance, include materials obtained directly from asource, such as a sample of whole blood, as well as materials pretreatedusing techniques, such as filtration, precipitation, dilution,distillation, mixing, concentration, inactivation of interfering agents,etc. These steps may be carried out prior to the sample being introducedto the cartridge or may be carried out by the cartridge itself.

The sample may be introduced prior to the cartridge being inserted intothe reader or after the cartridge has been inserted into the reader. Thecartridge may be so designed that the sample is introduced by way ofcapillary action.

The analyte to be detected can be any desired analyte and may includeproteins, peptides, antibodies, nucleic acid, microorganisms (such asbacteria and viruses), chemical agents, toxins, pharmaceuticals,metabolites, cellular moieties and the like. For example, the presentsystem may be adapted to detect any type of analyte that can bind asuitable binding agent. The binding agent may be any suitable agentwhich is able to bind specifically to the analyte to be detected. Forexample, if the analyte is a protein or peptide, the binding agent maybe a receptor or antibody which is capable of specifically binding tothe protein/peptide. Conversely an antibody may be bound by aprotein/peptide which the antibody is designed to specifically bind to.Nucleic acids may be bound by other nucleic acids which are capable ofspecifically hybridising to the analyte nucleic acid. Microorganisms maybe bound by antibodies which specifically bind to proteins on thesurface of the microorganism. Chemical agents, toxins, pharmaceuticals,metabolites may be bound by chemical moieties which are capable orreacting or binding to the aforementioned chemical analytes viaappropriate bonding reactions, or affinities. Many types of bindingtechniques are well known to those of skill in the art.

Moreover, the binding agent may be an enzyme or an enzyme substrate. Forexample analytes such as glucose through well described enzymaticmethodologies may be detected, for example the reaction product formedfollowing the enzyme reacting with the glucose may be detected by usingelectrochemical, or optical detection techniques known to the skilledaddressee. Such measurements can be made as standalone measurements orin combination with other analytes to be detected in the sample.

The binding agent is attached to a magnetic agent, such as aparamagnetic particle by methods including physical adsorption, covalentchemical coupling, non covalent chemical bonding (e.g. biotin-avidin) orany combination of these. In a preferred embodiment, the binding agentis bound to the paramagnetic particle via non covalent chemical bonding(e.g. biotin-avidin association). The paramagnetic agents/particleswhich are functionalised to comprise the binding agent bound thereto,may simply be deposited within a channel of the cartridge, such thatupon the sample being applied to the cartridge and being drawn into thechannel(s), the functionalised paramagnetic agents/particles areresuspended by the fluid sample and hence come into contact with anyanalyte in the sample.

As mentioned above as well as the binding agents, the cartridge maycomprise one or more further reagents deposited within said microfluidicchannels(s), which reagents may facilitate detection of the capturedanalyte. For example said one or more reagents may include a label whichhas been adapted to specifically bind to the captured analyte, thusfacilitating its detection. These reagents may be deposited separatelyor in combination with other reagents, such as functionalisedparamagnetic particles.

Further reagents deposited within said microfluidic channel(s) may havefunctions including but not limited to improving stability of otherreagents, improving/controlling resuspension of reagents, preventingcoagulation of blood sample, providing a substrate or cofactor requiredfor a enzymatic or chemical reaction, controlling the pH or ionicconditions of sample once applied to the cartridge, enhancing the signalproduced by the label, and enzymes which convert the product from alabel-induced reaction to a species to be detected by the reader.

Bound analyte may be detected indirectly providing the bound analyte iscapable of generating a detectable signal, whereby removal of boundanalyte from the detection area will result in a decreased signal, orupon binding of the analyte a reaction may take place, so as to generatea reaction product and after removal of bound analyte from the detectionarea, the decreased amount of unreacted analyte may be detected.However, in a preferred embodiment, bound analyte is contacted with alabel which is able to bind the bound analyte and a label/bindingagent/analyte complex is subsequently removed from the detection areaand the free unbound label that remains is detected. Typically the labelis able to bind to a different portion of the analyte to which the firstbinding agent binds, or is capable of binding to a region of the bindingagent/analyte complex which is formed only on generation of such acomplex.

Desirably the binding agent and any detection agent/label are in a drystate when deposited in the channel(s) of the cartridge.

When a capture agent and label (which are designed to facilitate captureand detection of the analyte) are desired, they can be depositedtogether or separately so they only come together upon rehydration withapplication of sample. Any other reagents which are desired for theassay can be deposited together with the capture and/or label orseparate to either or both of these. In this way, rehydration ofreagents can be ordered.

Each cartridge may be designed to carry out single analyte detection ormultiple analyte detection. Moreover, each cartridge may comprise morethan one microfluidic channel system, so that more than one assay may becarried out using a single cartridge.

Desirably the cartridges may easily be mass produced.

Once the cartridge has been loaded with a sample, any captured analytemay be detected indirectly by way of a suitable reader. The presentinvention provides such a reader and an important aspect of the presentinvention is that no buffer or additional fluid is required other thanapplication of sample to the cartridge. One advantage of this is thatthe cartridges themselves may be initially “dry”, that is contain littleor no fluid within the cartridge prior to sample application. This notonly simplifies manufacturing of the cartridges themselves, but alsoimproves shelf-life and allows many of the cartridges of the presentinvention to be stored at room temperature, with little degradation ofthe chemical or biological components within the cartridge prior to use.

In a further aspect there is provided a method of conducting an assay ona sample, the method comprising:

-   introducing a sample into a microfluidic cartridge of the present    invention such that any analyte present in the sample is capable of    being bound by a binding agent; and-   detecting a level of label (both bound and unbound to analyte), in    order to obtain a first reference/control value;-   removing bound analyte from the detection area; and-   detecting any unbound or unreacted analyte that remains after    binding of analyte present in the cartridge, or detecting a label    which is capable of binding the unbound or unreacted analyte.

Typically, the analyte/binding agent complex and analyte/bindingagent/label complex is capable of being removed or transported toanother location in the cartridge, in order that the unbound and/orunreacted analyte or unbound label may be detected.

In a further aspect there is provided an assay system for conducting anassay on a fluid sample, the assay system comprising:

-   -   a) a microfluidic cartridge according to the first aspect and        comprising paramagnetic particles (or preferred embodiments        thereof); and    -   b) a reader device, the reader device comprising:    -   i) a receiving port for introducing the cartridge into the        reader;    -   ii) a magnet or magnetic force generating means, capable of        applying a magnetic force to the cartridge, so as to be able to        remove the paramagnetic particles from the detection area within        the cartridge; and    -   iii) detection means for detecting any label present within the        detection area of the cartridge both before and following        removal of an analyte/binding agent complex from a detection        area of the cartridge.

Unlike other systems, the present invention is based on the removal ofspecifically bound complex from the detection area. A first signal maybe detected following reconstitution of the label and/or binding agent.The label may be present in excess, so its initial signal is at amaximum or near maximum level. However, upon complex formation andremoval of a label/analyte/binding agent complex, from the detectionarea, such as by application of a magnetic force (if, for example,functionalised paramagnetic particles are used as a capture phase), adecrease in the signal may be detected which is inversely proportionalto the amount of analyte present in the sample and hence which wascapable of binding to the label and binding agent.

The reader includes a receiving port into which the cartridge is to beinserted. The reader may be adapted so as to ensure correct insertion ofthe cartridge and this could take a variety of forms. For example, thecartridge may be initially located on a carrier mechanism which entersthe reader, such as may be found in computers for loading CDs and thelike. Alternatively the receiving port may be sized to allow thecartridge to be received and an internal stop member may be found withinthe reader which the cartridge abuts once inserted correctly.Additionally, or alternatively, features found on or cut into thesurface of the cartridge may be designed to co-locate with featuresfound within the reader and only once the cartridge is correctly locatedin the reader, will the cartridge be able to be read.

In a further aspect the cartridge is preloaded into the reader, and maybe locked in place, forming a combined single-use cartridge and reader.In this aspect the meter and cartridge are not separable and thereforethe meter and cartridge can be used once only as a single integrateddisposable device.

In the embodiment where the binding agent is bound to the surface ofmagnetic agents, such as paramagnetic beads, it is understood that thereader will comprise a permanent magnet or electromagnet which isdesigned to apply a magnetic field or be brought into close proximity ora magnetic field applied, in order to hold the paramagnetic particles ina particular area of said microfluidic channel of the cartridge. Thisarea may be spacially located away from the detection area.Concentrating the paramagnetic particles into a particular area out withthe detection area may serve to facilitate detection of any analyte bymeasurement of unbound label or unbound analyte and/or increasesensitivity of detection. The permanent or electromagnetic field may bereduced or increased, such as by moving a permanent magnet closer to, orfurther away from the cartridge, or by increasing or decreasing theintensity of the applied field.

In use the magnet or magnetic field may be used to pull paramagneticparticles away from the detection area.

In one embodiment, paramagnetic particles are used to capture analyteand a label used for detection. After a period of time to allowformation of paramagnetic particle-analyte-label complexes to form, amagnet, or magnetic field may be used to pull the paramagnetic particles(including paramagnetic particles complexed to analyte and label) awayfrom the detection area allowing measurement of analyte concentration bythe decreasing concentration of label as analyte concentrationincreases. In this embodiment, the remaining label that is detected inthe detection area is any label that is not part of a fully formedreaction complex (that is, is not bound to a paramagnetic particle).This could be unbound free label or label bound to analyte.

The label used for detecting analyte is deposited in such a way so thatupon rehydration by cartridge filling by application of sample the label(and other reaction components) is distributed throughout the detectionarea. The initial signal generated by the label is therefore independentof analyte concentration, and can form an important baseline for thespecific measurement which is made after removal of label that has beenbound to the capture phase reagents (such as functionalised magneticparticles) via specific analyte binding.

The reader of the present invention further comprises detection meansfor detecting any captured analyte within the sample cartridge. Thedetection means may be any suitable means depending on the particularassay. For example, the detection means may be a potentiostat, which maybe used to detect an electrochemical signal, once generated by thelabelled or unlabelled bound analyte or reaction product. The boundanalyte/reaction product may have intrinsic electrochemical propertiesand may be measured, for example, by current generated after anappropriate potential has been applied, or a further label may be usedto separately bind the bound analyte and the label detected byelectrochemical means. Other labels which may be employed and hence thedetection means adapted accordingly, include fluorescent labels,radiolabels, phosphorescent labels, colloidal metal particles,bioluminescent labels, colourimetric labels and the like. Moreover, asmentioned above the bound analyte or radiation product itself may bedirectly detected using techniques such as Raman spectroscopy and thelike. Also, measurements of absorbance of naturally occurring componentsof the sample, or species generated by a chemical or enzymatic reactionor label could be used.

When the reader uses electrochemical detection the methods of signalmeasurement include, but are not limited to chronoamperometric,potentiometric, impedance, linear sweep, charge transfer, potentiometricstripping, galvanometric, voltametric analysis (differential pulse,square wave, sample DC, normal pulse, AC voltammetry, AC secondharmonic, differential normal pulse) and the like.

The detectable labels may be used alone, or in conjunction with amicroparticle or bead, such as a metal oxide, polysaccharide or latexparticle. Many types of latex and other particles are known in the art

The reader may include other features, such as a heating device to allowassays to be conducted at a particular temperature, as well asappropriate electrical circuitry and software to allow the reader to beprogrammed to carry out one or more different assays.

The platform system of the present invention, comprising cartridge andreader provides a number of distinct advantages:

-   -   1. Reduced Sample Volume: capillary introduction of a fluid,        such as a finger stick blood sample, reduces the complexity for        the user and allows the tests to be performed in any environment        (e.g. ambulance, point of care, doctor's surgery, battle field,        home etc), and similar to glucose testing, allowing products to        be placed anywhere.    -   2. Room Temperature Stability: Many existing IVD tests require        refrigerated storage and shipping, this requirement adds        significant cost to the product and also restricts the usage and        distribution of the product. The initial “dry” nature of the        sample cartridges aids in their stability and shelf-life. In        addition, the specific assay method described in this patent        allows for an initial background measurement before the removal        of specifically bound complex from the detection area. This        initial background measurement can be used for correction        purposes.    -   3. Low material costs and a simple manufacturing process allow        for low costs of goods (COGs), allowing substantial and        increased profits to be generated by the sales of IVD strips.        This is especially needed in the immunoassay and molecular IVD        market where the conventional tests tend to be of high        complexity driving both the strip material costs and overall        assay cost higher.    -   4. Low cost of reader. No need for a wash buffer which        simplifies reader mechanics. Use of simple detection methods        such as, electrochemical or simple optical measurements, allows        the reader instrumentation to be kept simple and therefore at        low cost also.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described by way of exampleand with reference to the figures which show.

FIG. 1 shows a schematic representation of a sample cartridge inaccordance with the present invention;

FIG. 2 is a schematic representation of how a cartridge of the presentinvention may be formed;

FIGS. 3 and 4 are photographs of a cartridge according to the presentinvention showing various features;

FIG. 5 is a schematic and schematic view of a reader device inaccordance with the present invention;

FIG. 6 shows a schematic representation of the reader internalmechanisms;

FIG. 7 shows graphed experimental results for the response of total PSAsubtraction assay carried out with wet reagents in a test cartridge inaccordance with the present invention;

FIG. 8 shows graphed experimental results for the response of an assayfor free PSA carried out with dried reagents and fluorescent detectionin a test cartridge in accordance with the present invention;

FIG. 9 shows the instrument functionality block diagram in accordancewith the present invention;

FIG. 10 shows a schematic representation of an example homogeneousimmunoassay format in accordance with the present invention;

FIG. 11 shows a reaction mechanism of a desired embodiment of thepresent invention.

FIG. 12 shows a schematic of the disposable cartridge including 20measurement channels in accordance with the present invention;

FIG. 13 shows a photograph of a physical embodiment of a disposablecartridge including 20 measurement channels in accordance with thepresent invention;

FIG. 14 shows a schematic of a disposable cartridge including a singlecommon counter electrode in accordance with the present invention;

FIG. 15 shows a photograph of a physical embodiment of a disposablecartridge including 4 measurement channels in accordance with thepresent invention.

FIG. 16 shows a typical graphed GOD titration curve whereby the currentvalue plotted is extracted from the 3 second point of a 3 secondchronoamperometric transient after the GOD was allowed to react with thesubstrate system for 2 minutes;

FIG. 17 shows a graphed GOD titration whereby the current value plottedis extracted from the 3 second point of a 240 second transient;

FIG. 18 shows a graphed GOD titration measured at −350V after a 2 minreaction following a 4 min incubation at an applied potential of −350mV;

FIG. 19 shows a graph comparing GOD titration curves for a normal 2minute GOD titration and a 2 minute titration after the applied −350 mVpotential for 4 minutes;

FIG. 20 shows a graph of the uncorrected post magnetic separationmeasurement of PSA concentration in accordance with the presentinvention;

FIG. 21 shows a table of the difference between post magnetic separationcurrent and the pre magnetic separation current for all concentrationsof PSA in accordance with the current invention;

FIG. 22 shows a graph of the difference between post magnetic separationcurrent and the pre magnetic separation current for all concentrationsof PSA in accordance with the current invention;

FIG. 23 shows a table of the ratio of the post magnetic separationcurrent to the pre magnetic separation current in accordance with thecurrent invention;

FIG. 24 shows a graph of the ratio of the post magnetic separationcurrent to the pre magnetic separation current in accordance with thecurrent invention;

FIG. 25 shows a graph of the difference between the mean pre magneticseparation current and the post magnetic separation current inaccordance with the current invention; and

FIG. 26 shows a graph of PSA concentration measured by dividing the premagnetic separation measurement slope by the post magnetic separationmeasurement slope, in accordance with the current invention.

A sample cartridge (14) in accordance with an embodiment of the presentinvention is shown in FIG. 1. A fluid such as blood is applied to thesample introduction port (13) (via, for example, finger or venousblood). In this particular embodiment four channels (4,5,6,7) span fromthis one sample introduction port (13). Although not to be construed aslimiting, the further description will relate to the sample being asample of whole blood.

The blood is applied to the sample application port (13), the initialsingle channel splits into 4 separate channels. Each channel allows ameasurement to be made, this could be 4 measurements of the same analyteresulting in replicate results of a single analyte or a differentanalyte could be measured in each channel. A 4 channel version of thestrip is shown in FIG. 1. A 20 channel strip (105) is shown in FIG. 12,with a photograph of a physical embodiment (106) of the strip with 15channels shown in FIG. 13. In FIG. 12 a close up of the electrodeconfiguration shows a counter electrode (107), a working electrode (108)and relevant reagents (109) deposited homogenously, such that thedetection zone (108) is located within the area of the channel where theanalyte binding by specific reagents occurs. An alternative embodimentin FIG. 14 shows a strip design (110) where a common counter electrode(111) is utilized as opposed to having a separate counter electrode foreach working electrode. This common counter electrode allows theelectronics in the reader to be simplified and also allows thedisposable cartridge size to be reduced as the disposable cartridgewidth is influenced by the total number of electrodes. With a separatecounter electrode for each working electrode, the total number ofelectrodes is 40 for a cartridge capable of 20 working electrodemeasurements, while for a cartridge with a common counter electrode, thetotal number of electrodes is reduced to 21.

The total sample application may be as small as less than 1 □l dependingon the number of channels to fill therefore when the user applies asample, such as a drop of blood, all channels (4,5,6,7) will fill undercapillary force. This process is very fast and more in tune with bloodglucose strip filing as opposed to the lengthy blood separation fillingof some immunoassay platforms. Deposited in the four channels (4,5,6,7)are paramagnetic particles functionalised with antibody and labelfunctionalized with antibody and label enzyme, as well asanti-heterophile reagents, stabilising agents, electrochemical mediatorand anticoagulant (8,9,10,11). Deposited in the sample introduction port(12) is enzyme substrate which is converted to an electrochemicallymeasurable species by the label enzyme (13) to produce a measurablesignal for detection by the reader.

The electrodes present in each channel (1,2,3) are used to make theelectrochemical measurements, however in other examples optical or othermethods of detection measurements could be made. The electrodes are alsoused to tell the user when enough blood has been applied to thecartridge.

When the cartridge (14) is formed from three substrates (20,21 and 22)as shown in FIG. 2 the channels are formed by removal of material froman adhesive layer (24), sandwiched between a base layer (20) with screenprinted electrodes (23) and a lid layer (22). In FIG. 2, the lid (22) isshorter than the channel formed in the adhesive (24) which forms an airvent where the edge of the lid (25) meets the channel (24), when thecartridge is assembled. This allows the cartridge to fill by capillaryforce, and also forms a fluidic stop feature. FIG. 15 shows a photographof a physical embodiment of this strip design (112). In this particularembodiment there is a circular aperature (113) cut in the top lid toallow the filing of the device by applying a drop of sufficient volumeof sample into this circular area.

As the cartridge (14) is inserted into the reader, a cartridge heatingmechanism may be initiated, heating the cartridge to a predefinedconstant temperature for the duration of the test.

In each of the four sample channels (4,5,6,7) on the cartridge there maybe electrodes (1,2,3 in FIG. 1). Through the reader, checking theelectrical continuity between the electrodes, the reader will be able toconfirm that the channels (4,5,6,7) have been successfully filled withsample. This can be performed through a simple conductance measurement.For a specific channel, if the electrodes (1,2) have been successfullywetted with blood (meaning that the channel (4) has been filledcompletely with sample) then an electrical current can conduct from oneelectrode to the other through the blood sample. Otherwise if the bloodsample is not present, or has only partially filled the channel, then atleast one of the electrodes will not be wetted, meaning the electricalcurrent cannot flow from one electrode to another. Additional electrodescan be added to each channel for more accurate determination of devicefiling (to ensure sample has completely covered the electrodes used inanalyte detection). An additional electrode positioned downstream (inrelation to the flow of sample upon addition to the cartridge) of theother electrodes in each channel would satisfy this. The current flowbetween these additional electrodes in different channels would thenindicate that those channels were both full, or lack or current flowwould indicate that one or both channels were not full of sample.

In the present cartridge/assay system, it shall be possible to measurethe hematocrit of the blood sample.

FIG. 3 shows a photo of a single channeled cartridge (41) with thechannel filled with fluid (33) up to the end of the channel which hasbeen defined by the edge of the lid (37) producing an air vent (32)which allows the device to fill by capillary force and forms a fluidstop feature.

The inner surfaces of the channel are required to have some hydrophilicproperties to allow filling by capillary force. In one embodiment twohydrophilic surfaces are utilised however alternative combinations ofhydrophilic/hydrophobic surfaces could be used to fill the strip bycapillary action

In FIG. 3 the sample has been applied to the sample introduction port(33) and fills the channel formed by the adhesive layer (31), withchannel edges indicated (36), between the base layer (30) with printedelectrodes (39,40) and the lid layer (34). The electrodes are leftexposed (39, 40) as the adhesive layer stops before the end of thedevice (38), as does the lid layer (37). This allows the electrodes tobe connected to the reader. This can be seen more clearly in FIG. 4which shows an angled side view photograph of the same single channeldevice as depicted in FIG. 3. In FIG. 4, the base layer (52) withprinted electrodes (56) is partially covered by an adhesive layer (51)where the edge can be clearly seen (54). The adhesive layer (51) ispartially covered by a lid layer (50), where again the edge is visible(53).

As the blood fills the sample channel (33) (see FIG. 3) thepre-deposited dried reagents are resuspended by the blood, therebyallowing binding any analyte/s present. Potential positioning of driedreagents are shown in FIG. 1 (8,9,10,11,12). The blood fills the channel(33) to the stop features (37), see FIG. 3. Once the functionalisedcapture and detection reagents are resuspended, incubation with theblood sample would be allowed to occur for a defined period of time(incubation time) and controlled by appropriate software and programmingof the reader. Paramagnetic particles may be chosen as the capture phaseand particles (eg latex) or conjugates chosen as the label, or detectionphase due to their high mobility and functionality (size dependent i.e.diffusion coefficients etc) to reduce diffusion distances and ultimatelyincubation time. This type of reaction will be very efficient andreproducible at binding analyte from blood samples. During the captureand detection phase binding of analyte, a hematocrit measurement mayperformed by electrodes (39,40). The hematocrit value can be used by thereader to calculate the final concentration of the analyte as thereference value may be a plasma measurement made by a clinical analyser.A hematocrit measurement may be required to correct for theconcentration difference associated with analyte present in a givenvolume of sample due to differing ratios of red blood cells to plasma.Therefore a whole blood measurement may be corrected for this differenceby means of a hematocrit measurement so that results are consistent withthose associated with a plasma sample.

After the capture phase and detection phase reagents have bound anyanalyte in the blood a permanent magnet or electromagnetic field can beused to remove the paramagnetic particles (including paramagneticparticle-analyte-label complexes) from the vicinity of the workingelectrode (In FIGS. 3, 39 and 40 represent the counter/referenceelectrode and working electrode where either electrode could beconfigured through the reader for either function). In this case,detection of the remaining label (either directly or through alabel-catalysed or label-mediated reaction) can then be detected byelectrochemical means at the working electrode.

It should be appreciated that the foregoing description, with referenceto FIG. 1, has been made in relation to a four channel cartridge, and inrelation to FIGS. 3 and 4 a single channel cartridge, but the presentinvention also relates to multi-channel e.g. 6, 7, 8 etc cartridges,with an example of a 20-channel cartridge shown in FIGS. 12, 13 and 14.Each channel may carry out the same reaction forreproducibility/accuracy purposes, or may be designed to carry outdifferent assays—in this way each cartridge may be capable of carryingout a “multi-plex” reaction.

Ultimately a measurement is made (e.g. electrochemical) by a readerusing electrical, optical or other detection means, suitable for thelabel to be detected. For example, if the label is an enzyme whichcatalyses a reaction which generates a product that is electrochemicallyactive, the detection means may be electrical, via electrodes within thecartridge, see FIG. 3 (39,40). Schematic views of a hand held reader inaccordance with the present invention are shown in FIGS. 5 and 6. Thereader (70) comprises a platform (72) for receiving and holding acartridge (61) of the present invention. There is also provided suitabledetection means; a PCB (103) comprising a connector to interface withthe cartridge (104), electrical circuitry and an associated computerchip or chip(s) and software for controlling the reader and conductingthe assay. In addition because the described system has the flexibilityto perform assays using paramagnetic particles, the meter has thefunctionality to move a permanent magnet (101) via a motor (102) in theproximity of the cartridge. For example, towards the cartridge and awayfrom the cartridge, or in any other orientation in order to manipulatethe paramagnetic particles as required.

The reader can also control the temperature of the sample applied to thecartridge by means of active heating in the case where the cartridgeplatform (72) comprises of a material that is conducive to conductingheat

FIG. 5 shows envisaged physical embodiments of the meter where a resultcan be displayed on the reader screen (62).

The primary functions of the reader are described in FIG. 9, the readerfunctionality block diagram, and are as follows:

-   -   1. Cartridge Signal Measurement    -   2. Cartridge Temperature Control    -   3. Paramagnetic Particle Manipulation    -   4. Calibration Mechanism

Cartridge Signal Measurement: The reader shall be capable of exposingeach of the separate test channels to separate precise electricalsignals in order to commence, maintain and control the requiredelectrochemical reaction that is to be conducted within the particulartest channel of the test cartridge. Examples of such electrical signalsare steps from one voltage potential or from an open circuit state toanother voltage potential state, a linear sweep from one potential toanother in a defined time, or the creation of a non linear waveform forexample a sine wave of a particular frequency and voltage magnitude. Thereader may use analogue switching multiplexers in order to switchbetween different measurement channels.

The Instrument shall be capable of measuring theelectrical/electrochemical response from each test channel separately.Examples of the signals to be measured from the test cartridge aredirect or alternating currents or a voltage potential. The reader mayuse analogue switching multiplexers in order to switch between differentmeasurement channels.

The reader shall have a connector that makes physical contact with theprinted electrodes on the test cartridge to ensure the delivery ofelectrical signals to and from the cartridge. This connector could bemounted directly on to the PCB of the reader.

As the proposed system comprises of a cartridge and reader with theability to perform multiple measurements, for example 20 separatemeasurements on a single applied test sample, the system is capable ofusing these multiple measurements to produce improved results withregards to accuracy and reliability. In the instance where the testsystem is used to measure multiple separate measurements of the sameanalyte, the system would be able to perform a mean or truncated meananalysis of the obtained 20 results. For truncated mean analysis, thiswould involve the instrument software sorting the 20 results in order ofmagnitude, removing a number of the highest and lowest results (e.g. 5)and taking an average of the remaining (e.g. 10) results. The benefit ofthe truncated mean over a standard mean is that results that arestatistical (or practical) outliers can be removed from the data setbefore the mean is taken. This removes the risk of abnormal resultsshifting the resultant mean substantially higher or lower than theexpected result.

In the case of a low cost assay system the specific improvementsassociated with this methodology include the ability to remove resultsthat are abnormally high or low due to factors such as incorrect testsample volume, incorrect reagent deposition volume, incorrect reagentformulation, and physical or environmental damage to the test samplechannel or associated reagents. The system discussed in this patent isparticularly suited to this truncated mean analysis as each of thesample channels in the test cartridge are truly separate including aseparate reagent deposition, a separate detection means (e.g a workingand counter electrode) and a separate sample channel.

The system could be configured to measure multiple measurements ofmultiple analytes and therefore improve the accuracy of the results formultiple analytes within a single test.

Cartridge Temperature Control: The reader shall be capable ofcontrolling the temperature of the test cartridge. The mechanical designof the reader shall result in the instrument PCB being in contact withthe test cartridge. Therefore it would be possible to have the heatgenerated from a source (for example a high wattage low value resistor,a MOSFET transistor or a thin film flexible printed circuit boardheating element) coupled into the cartridge through the instrument PCB.This could be achieved by having a bared copper area of the PCB incontact with both the test cartridge at one point, and the heat sourceat another point.

Alternatively the reader design could comprise of a separate heatingblock made of suitable material for heat transfer to the test cartridge,such as aluminium. This separate heating block could be place within thereader such that it is in direct contact with the test cartridge.

The control of the heating of the test cartridge shall come by placingone or more temperature sensors in contact with the surface that is indirect contact with the test cartridge. The reader can then monitor thetemperature that the test cartridge is being exposed to and adjust theamount of heat energy being transferred to the cartridge accordinglythrough controlling the heat source. (For example decreasing orincreasing the amount of current flow through a MOSFET).

Paramagnetic Particle Manipulation: The reader shall be capable ofgathering the paramagnetic particles contained within the test channelsof the test cartridge to a pre defined position. For example, the readermay gather the paramagnetic particles on the cartridge surface oppositethe test channel working electrode, or it may gather the paramagneticparticles to another location on the test channel specially separatedfrom the detection area. One method of achieving this would be to use apermanent neodymium magnet to gather the paramagnetic particles. Amechanism could be implemented where the magnet can be physically movedin a single plane such that at one point in the mechanism movement it isin physical contact with the cartridge, meaning that the paramagneticparticles are gathered by the magnet, and at another point there is aphysical distance between the magnet and the test cartridge, meaningthat the paramagnetic particles are not under the influence of themagnetic field associated with the magnet.

The physical movement of the magnet in one plane could be motorisedthrough the use of, for example, a linear actuator, or through arotational motor using a gearing system, or through a clockworkmechanism, or through a mechanically sprung mechanism, or through amanual mechanism whereby the movement of the magnet is initiated andcontrolled by the user of the instrument.

In a different embodiment, the permanent magnet could be replaced by anelectromagnet, in which case the control of the paramagnetic particlescould be performed by energising or de-energising the electromagnet.Alternatively the reader could implement a stationary permanent magnetin a fixed position where the paramagnetic beads are to be gathered.

Using these different methodologies, the reader could affect theparamagnetic beads in such a way as to collect them to a particularlocation associated with the cartridge from their previously scatteredstate. This location could result in the paramagnetic beads being movedfrom one surface of the cartridge to another or along a surface of thecartridge, or between the cartridge surfaces or a combination of any ofthese.

Calibration Mechanism: Due to the variation in processes associated withthe manufacturing of disposable assay test cartridges it is normallyrequired for each batch of cartridges to be characterised and forspecific calibration values to be entered into the reader so that theassay response generated by the test cartridge can be normalised by thereader internally before the final assay result is reported. In order toovercome this issue the reader could have different sets of calibrationparameters pre loaded into its memory. The reader would know what set ofcalibration parameters to use for a particular test cartridge due to thevalue of a surface mount resistor that could be mounted either on thetest strip itself or on a separate substrate that can be inserted intothe meter independently of the test strip and in a different location.The instrument could then measure the value of the surface mountresistor which would be related to a specific set of pre loadedcalibration parameters. Set bands of resistances could relate to setcalibration codes. Alternatively the resistor could be replaced with acapacitor, and the reader could attribute different levels ofcapacitance with a calibration code. Alternatively, the resistor couldbe replaced with an inductor, and the reader could attribute differentlevels of inductance with a calibration code. Alternatively, acombination of any resistors capacitors and inductors could be used.

It is a physical feature of assay development that the ambienttemperature can influence the magnitude of response. In the presentinvention, this temperature effect will be driven through the effect oftemperature on diffusion, whereby an increase in temperature can resultin increased binding efficiency between the paramagnetic beads and thetarget analyte, and the label and target analyte. Temperature effects onenzyme activity will also make a significant contribution to the assayresponse where enzymes are used. The present system may be used, forexample in a doctor's office and home use, and the range of temperaturesthe system may be exposed to will be broad, from perhaps as low as 10degrees Celsius to as high as 40 degrees Celsius. One method of removingthis temperature effect is in the heating of the test strip to apredetermined temperature, for example 40 degrees C. This would removeany variation associated with the assay due to temperature effects.Heating the cartridge also helps to minimise any blood to blood effectsdue to differences in viscosity. Thus, the reader may also comprisetemperature control means, such as a heater as previously described.

In one particular embodiment, the sample cartridge and associated readerare designed for carrying out an immunoassay, where the analyte to bedetected is an antigen and the binding agent is an antibody.Paramagnetic particles may be functionalised by attachment of antibodiesagainst either free or free and complexed antigen.

Although not to be construed as limiting, the further description willrelate to the sample being a sample of whole blood and label detectionbeing enzyme driven electrochemical detection, and a paramagneticparticle capture phase.

As the blood fills the sample channels of the cartridge, the reagentswhich are pre deposited in the initial channel (12) see FIG. 1, andwithin each of the channels (8,9,10,11) as dry reagents, are resuspendedby the blood and start binding the analyte/s. The deposited reagentswithin each channel (8,9,10,11) contain paramagnetic particlesfunctionalised with an anti-analyte antibody, a label (e.g. ananti-analyte antibody-enzyme conjugate), electrochemical mediator,anti-coagulant, anti-heterophile reagents and stabilisation reagents. Incomparison the reagents deposited in the initial feed channel (12)contain the enzymatic substrate.

As the blood fills the strip the dried enzymatic substrate isresuspended and is transported by the blood into the 4 measurementchannels (where the enzyme substrate is in excess). The other reagentsin each channel are resuspended as the blood fills each channel. Theanti-analyte antibody-enzyme conjugate immediately starts convertingsubstrate and reacting with the electrochemical mediator. Theelectrochemical mediator is converted from the reduced form to theoxidised form. At the same time the analyte is being bound by theimmunoassay binding reagents (paramagnetic particles functionalised withan anti-analyte antibody and an anti-analyte antibody-enzyme conjugate(label)). These homogenous events which are occurring throughout thechannels are allowed to occur for a defined period of time (bindingtime, such as 2-4 minutes). These processes and the following processesare occurring in all 4 channels, it could be for the same analyte or fora different analyte in each channel whereby the antibody pairing wouldbe changed.

An electrochemical measurement is initiated at a defined time; thiscould be at the end of the binding time, during the binding time orimmediately as the strip is filled. A chronoamperometric measurement ismade whereby the current is measured at the electrochemical reductionpotential of the mediator (the electrochemical mediator is beingconverted to oxidised form by the label (free and bound)). At this pointall concentrations of analyte will have the same rate (current per sec)calculated from the chronoamperometric transient.

After the defined binding time a magnet (permanent or electromagnet) isapplied to the lid surface opposite the electrodes surfaces. Theparamagnetic particles are pulled via magnetic attraction to the innerlid surface. The working electrode is only measuring the sampleimmediately above it, therefore when the paramagnetic particles aremagnetically pulled to the inner lid surface opposite the electrodesthey will contain a mixed paramagnetic particle population. Theparamagnetic particle population will be split between paramagneticparticles with no analyte bound, paramagnetic particles bound toanalyte, and paramagnetic particles bound to analyte and label (fullyformed sandwich immunoassay complex).

The number of sandwich immunoassay complexes formed will be proportionalto the concentration of analyte in the blood sample. The higher theanalyte concentration in the blood the more immunoassay complexes formedand hence removed from the vicinity of the working electrode during themagnetic accumulation. This process is therefore changing the enzymelabel concentration at the working electrode and is occurring in ananalyte concentration dependant manner. This process is measured in realtime; therefore the rate calculated from the chronoamperometrictransient is proportional to the enzyme concentration which isproportional to the analyte concentration. The rate therefore decreasesas the analyte concentration increases (the more enzyme bound the moreimmunoassay complexes are formed). The assay format is therefore not aconventional heterogenous assay format but more in tune with ahomogenous assay format. This assay format is summarised in FIG. 10,specifically for an enzyme label using electrochemical detection.

FIG. 10 is a schematic of the homogeneous immunoassay format, with eachimage (A and B) representing schematically a cross section through thechannel in a cartridge, with the channel top (84) and bottom (85)indicated and shows the working electrode (81) before (FIG. 10A) andafter (FIG. 10B) the presence of the magnetic field. In A, labelparticles (82) and paramagnetic particles (83) are rehydrated by sample(87). The sample has also rehydrated enzyme substrate andelectrochemical mediator so the enzyme label is reacting with thissubstrate and mediator from the onset of rehydration. In A the bindingreaction is also occurring between the magnetic particles, analyte andlabel with fully formed immune complexes shown (86). Although only thearea above and around the working electrode is shown, this process isoccurring throughout the whole channel. The electrochemical measurementwould begin in A, after a defined period of binding time; the magnet(80) is applied to the top surface (84) opposite the electrode surface(84) as shown in B. All of the paramagnetic particles are accumulatedirrespective if an immune complex is formed or not. The amount of enzymelabel accumulated with the paramagnetic particles is dependant the onthe number of immune complexes formed which is proportional to theanalyte concentration. Because the paramagnetic particles and all fullyformed immune complexes are removed from the vicinity of the workingelectrode by the application of a magnet, as the analyte concentrationincreases, the electrochemically measured rate/response decreases.Although in this example the magnetic force is applied to the surfaceopposing the working electrode, the magnetic force could be appliedanywhere in the sample channel whereby paramagnetic particles areremoved from the detection zone.

In a desired embodiment of the present invention, the paramagneticparticle and label are used to form an immunoassay sandwich by capturingthe analyte, with the capture and label antibodies recognizing differentantigen epitopes. The desired embodiment of the current invention assayformat is to use glucose oxidase (GOD) as the label enzyme. GOD has beenextensively used for the measurement of blood glucose. Specifically anexcess of GOD and electrochemical mediator is dried in a strip, the GODreacts with the glucose contained in the blood, converting anelectrochemical mediator from the oxidised to reduced form which is thenelectrochemically measured at a electrode. This system works well as allthe reaction components are in excess so the glucose concentration isthe rate determining factor. GOD has been used as enzyme label for ELISAmeasurements however in electrochemical immunoassays GOD has been foundto be fairly insensitive as an enzyme label. This is driven from thefact that directly coupled electrochemical mediators have been employed.Oxygen is the true acceptor for the GOD reaction, but ferricyanide isused as an artificial acceptor to electrochemically couple the reactionso an electrochemical measurement can be performed. This reactionbetween ferricyanide and GOD is very unfavourable compared to oxygen andis overcome by very high ferricyanide concentrations. This problem isalso overcome in glucose sensors as GOD itself is in excess thereforeany inefficiency becomes insignificant. In an enzyme based immunoassaythe enzyme concentration is measured, therefore unfavourableinteractions between GOD and ferricyanide results in a sensitivityproblem. The desired embodiment of the present invention overcomes thisproblem by using a further coupled reaction as summarised in FIG. 11:The GOD coverts glucose and oxygen into gluconolactone and hydrogenperoxide respectively (using FAD as the cofactor of GOD). Horseradishperoxidase (HRP) and ferrocyanide are present in large excess (as isglucose), therefore any hydrogen peroxide produced will be immediatelyused by HRP to convert ferrocyanide to ferricyanide which is thenmeasured at the electrode with an electrochemical reduction reaction(this then electrochemically reduces the ferricyanide back toferrocyanide with the loss of electrons from the electrode). The GODbecomes much more sensitive as an enzyme label as it is no longer forcedto artificially react with ferricyanide, and its turnover of glucose isunhindered therefore lower GOD concentrations can be measured. This istrue of all enzyme labels; the enzyme rate for the natural reaction isgreater than any conceived coupled system whereby a measured event hasbeen forced into the enzyme reaction. Using an enzyme cascade allowsthis problem to be avoided as in the secondary reaction both the enzymeand the mediator are in excess and not rate limiting.

There are a number of advantageous to using the described scheme:

-   -   1. The GOD reaction is unhindered allowing sensitive        measurements of GOD concentration.    -   2. GOD is well known as a robust stable enzyme hence its use in        the blood glucose monitoring products. Other labels such as HRP        are known not be as stable, HRP instability in this embodiment        of the current invention can be overcome by enzyme redundancy        (vast excess).    -   3. An electrochemical reduction is performed to measure the        concentration of ferricyanide generated by the GOD/HRP reaction.        A reduction reaction will significantly reduce the influence of        electrochemical interferents in the blood. Most Interferents        interfere with oxidation reactions.    -   4. There are no linearity problems with respect to the        electrochemical measurement as all the components are in large        excess (due to high solubility) unlike some other HRP and other        enzyme substrates (such as alkaline phosphatase) where excess        cannot be achieved.    -   5. In most assays a background measurement is determined by        signal magnitude measurements during calibration. This        background measurement is an average value, normally with a        large associated error. The background can vary significantly        due to any variations in reagent concentrations deposited,        variations in detection areas, blood to blood differences etc.        This decreases the sensitivity of most assays. In this system        the background can be measured in each channel before the magnet        is applied and the specific signal measurement made. In this way        each measurement can be individually background corrected, which        allows for very sensitive and accurate assay results.

Due to the ability to have multiple channels within a cartridge, thereis a great opportunity to extend analyte measurement ranges. Forexample, typical immunoassay dose response curves are sigmoidal. This isdriven by either reagent saturation (insufficient reagent to maintainlinear binding) or saturation of the label/detection method (i.e. thedetection methods becomes saturated and can no longer measure the labelin a linear fashion). The present platform may however, allow a fulllinear response across the measurement range if required, and this canbe achieved by having different reagents, or reagent concentrations indifferent channels. For example, the reader can measure theconcentration of the label in one channel with reagents designed andmanufactured for very high sensitivity, but that have a limited range.Reagents in a second channel could be designed and manufactured to havelower sensitivity but greater range for the same analyte. Therefore, ifthe signal achieved in the first channel is over a threshold value (ieabove the measurable range of that assay set during calibration,representing a high analyte concentration) the signal from the otherchannel would be used and vice versa, if the signal from the secondchannel was below its threshold value (ie below its measurable range setduring calibration and representing a low analyte concentration) thesignal from the first channel would be used to determine analyteconcentration. The present platform allows different ways of achievinglinear responses across the measurable range, which will allow moreaccurate calibration resulting in better within and between sampleprecision resulting in better ATE. If a measurement were made before theparamagnetic particle complexes are removed from the detection area,this gives a background measurement which can be used to normalise theresults and therefore correct for any variations that may occur such aslabel concentrations or exposed detection area etc.

Multiple channels also provides the functionality to combine differenttests on the same cartridge where one channel could have reagentsdesigned for measurement of, for example, creatine kinase (CK), andanother channel with reagents designed for the measurement of, forexample aspartate aminotransferase (AST), and another channel designedfor the measurement of, for example hematocrit (Hct). Such a combinationof tests has great potential use in the veterinary field for use onblood samples, for example from horses. In the case of these analytes,CK and AST could be measured by a sandwich immunoassay using a schemesuch as that described in FIG. 10. Either or both could also be detectedusing their enzymatic activity producing detectable reaction productsthrough enzyme linked reactions. These tests could be combined on acartridge with a hematocrit measurement using conductance of bloodsample between two electrodes. Different analytes could also be detectedwithin a channel. For example, fluorescent labels with separateemissions could be functionalized against different analytes and usedwith magnetic particles functionalized against all analytes to bedetected in a reader with the detection capability to read the signalsproduced from all the different label emissions.

Here follows experimental data generated by a specific embodiment of thecurrent invention. The electrochemical detection of signal is made by acommercially available potentiostat, in place of the reader.

Electrochemical Assay of Total PSA (I)

Materials

Neutravidin: Thermo Scientific, Cat 31000 (Neutravidin biotin bindingprotein).

Maleimide-PEG2-biotin: Thermo Scientific, Cat 21901 (EZ-linkmaleimide-PEG2-biotin).

Latex particles: Invitrogen, Cat C37259 (CML latex, 4% w/v, 10 um)

Paramagnetic particles: Ademtech, Cat 03223 (200 nm Strep+ paramagneticparticles)

Antibody 1H12: Hytest, Cat 4P33 MAb 1H12 (Anti-PSA, human)

Antibody 5A6: Hytest, Cat 4P33 MAb 5A6 (Anti-PSA, human)

bGOD: Rockland, Cat B000-07 (biotinylated glucose oxidase)

PBS: Thermo Scientific, Cat 28372 (BupH phosphate buffered saline packs)

BSA: Sigma, Cat A4503-50 G (Albumin, from bovine serum)

Water: Sigma, Cat W4502 (water for molecular biology)

Ammonium acetate: Sigma, Cat A1542-250 G (Ammonium acetate)

MES: Sigma, Cat M8250-25 G (MES hydrate)

Fenocyanide: Sigma, Cat P3289-100 G (potassium ferrocyanide)

Glucose: Sigma, Cat G8270-1 KG (D-(+)-Glucose)

HCl: Sigma, Cat H1758-100 ML (hydrochloric acid, 36.5-38%)

NaOH: Sigma, Cat 72068 (sodium hydroxide solution)

HRP: BBI Enzymes, Cat HRP4C (Horseradish Peroxidase)

2MEA: Thermo Scientific, Cat 20408 (2-mercaptoethanolaminehydrochloride)

PSA standards (calibrated against WHO 1^(st) IRP (96/670)): PerkinElmer, Cat A073-301 (ProStatus PSA free/total kit, Delfia)

Biotin quantification kit: Thermo Scientific, Cat 28005 (Pierce biotinquantification kit)

Size exclusion columns: GE Healthcare, Cat 17-0851-01 (PD10 columns)

EDTA: Sigma, Cat EDS-100 G (ethylenediamine tetracetic acid, anhydrous)

Tween: Sigma P7949-100 ML (Tween-20)

DMSO: Thermo Scientific, Cat 20684 (dimethylsulfoxide)

Acetic acid: Sigma, Cat 32,009-9 (acetic acid)

Reagent Preparation

Antibody Biotinylation.

Antibody Disulphide Bond Reduction

Antibody 1H12 and 5A6 are reduced using 2MEA in 1 mM EDTA in PBS, at 37degC for 90 min. Reduced antibody is passed through a PD10 column andcollected in 1 mM EDTA in PBS and fractions found to contain protein (bymeasurement at 280 nm on UV spectrophotometer) pooled. The concentrationof reduced antibody is calculated using the extinction coefficient ofthe antibody of 1 mg/ml=1.4 absorbance units at 280 nm.

Binding of Maleimide-PEG2-Biotin to Antibody

Maleimide-PEG2-biotin is added to the reduced antibody in molar excessto allow efficient binding to occur and incubated for 3 hours at roomtemperature. This is then passed through another PD10 column which hasbeen pre-equilibrated with 1 mM EDTA in PBS, pH7.2. 500 ul fractions arecollected and measured using the UV spectrophotometer at 280 nm. Thefractions containing significant protein levels are chosen and combined.A sample of this solution is measured again at 280 nm by absorbance, andthe concentration of antibody determined using the extinctioncoefficient of the antibody of 1 mg/ml=1.4 absorbance units at 280 nm.The number of biotins bound per antibody are then determined using thePierce biotin quantification kit, according to the manufacturer'sinstructions.

Latex.

Neutravidin Adsorption

10 um latex is washed in MES buffer (50 mM MES, pH6.5) usingcentrifugation at 16100×g for 5 min at 4 degC to pellet the particles.The latex is resuspended at a concentration of 2% solids. Neutravidin isprepared at a concentration of 400 ug/ml in water. 2% latex and 400ug/ml neutravidin are added together in equal volumes and mixed well.They are incubated for 18 h with mixing on a rotary mixer (30 rpm) atroom temperature. The particles are then washed in an equal volume ofPBS (pH7.2) 3 times (using centrifugation at 16100×g for 5 min, 4 degC)and resuspended in the same at a concentration of 2% solids.

Biotinylated GOD and Biotinylated Antibody 5A6 Binding to Latex

10 um latex is washed in 50 mM ammonium acetate, pH4.2 (usingcentrifugation at 16100×g for 5 min, 4 degC) and resuspended in the sameat a concentration of 1% solids. Biotinylated GOD is diluted in 50 mMammonium acetate, pH4.2 to a concentration of 160 ug/ml. Biotinylatedantibody 5A6 is diluted to a concentration of 40 ug/ml in 50 mM ammoniumacetate, pH4.2. An equal volume of 40 ug/ml b5A6 is then added to 160ug/ml bGOD. This mixture is then combined 1:1 with 1% neutravidin coatedlatex. This solution is mixed well and incubated for 30 min at roomtemperature with shaking on a rotary mixer (30 rpm).

The particles are then washed 4 times (using centrifugation at 16100×g,5 min at 4 degC) with an equal volume of PBS, pH7.2 to remove anyunbound biotinylated GOD and biotinylated antibody and resuspended inPBS, pH7.2 to give a latex concentration of 1% solids.

Paramagnetic Particles

Binding of Antibody to Particle

200 nm streptavidin coated paramagnetic particles are washed (using amagnetic separator) in 0.1% tween in PBS, pH7.2 and resuspended in thesame to give a concentration of 0.5% solids. Biotinylated antibody 1H12is diluted in 0.1% tween in PBS, pH7.2 to give 50 ug/ml. An equal volumeof 0.5% paramagnetic particles and 50 ug/ml biotinylated antibody arecombined, mixed and allowed to incubate for 30 min at room temperature,with shaking using a rotary shaker at 30 rpm.

The paramagnetic particles were then washed 4 times (using a magneticseparator) in an equal volume of 0.1% tween in PBS, pH7.2 andresuspended in the same to give a concentration of paramagneticparticles of 1% solids.

Assay Procedure

Assay reagents were combined in the following volumes and concentrationsinto an eppendorf tube:

1% paramagnetic particles (with bound b1H12): 2 ul

150 mg/ml BSA, 0.25% tween all in PBS, pH7.2: 4 ul

1% Latex (with bGOD and b5A6 bound): 2 ul

PSA standard: 2 ul

All reagents added to eppendorf separately and kept separate in tube.All reagents finely combined by mixing upon addition of the:

Reaction buffer: 10 ul

(Reaction buffer comprises 1M MES, pH 6.0, 200 mM glucose, 200 mMferrocyanide, 2 mg/ml HRP)

These reagents are mixed thoroughly then 0.7 ul is added to a cartridge,which is connected up to a potentiostat (Autolab PGSTAT12). Thecartridge is made up of a single channel, spanned by 2 screen-printedcarbon electrodes (one working electrode and one counter/referenceelectrode) as shown in FIGS. 3 and 4. A measurement is made by asubtraction method, as follows.

Subtraction Assay

After 4.5 min incubation, the potentiostat changes from open circuit to−0.1V and the transient recorded with data points every 0.1 s. After anincubation of 5 min a magnet is applied to the cartridge in a way toremove the paramagnetic particle complexes from the working electrode(ie. on the opposite surface to the electrodes). The resultant transientis recorded for a further 5 min. The rate of reaction at the workingelectrode is calculated from the data obtained between around 200 s and300 s after the potential step to −0.1V.

Summary of Assay Mechanism

In this assay mechanism (as summarised in FIG. 11), as soon as thereagents are combined, GOD converts glucose to gluconolactone withconcurrent conversion of oxygen to hydrogen peroxide. The hydrogenperoxide then acts as a substrate for HRP which can then oxidiseferrocyanide to ferricyanide. The concentration of ferricyanide producedis therefore dependent upon the concentration of hydrogen peroxideproduced which is itself dependent upon the concentration of GOD. Theconcentration of ferricyanide is measured by chronoamperometry, in thiscase by measuring the current produced at a potential of −0.1V betweenthe working and counter/reference electrodes in a two electrode system.When the magnet is applied (away from the working electrode, on theopposite surface, directly above the working electrode) the paramagneticparticles will move to the magnet away from the vicinity of the workingelectrode. Depending upon the PSA concentration, some label particleswhich are coated in GOD will be removed also through their attachment toparamagnetic particles via PSA. In this case the concentration of GOD atthe working electrode surface (where the measurement occurs) will thendecrease in a manner dependent upon the number of label particlesremoved, which is dependent upon the concentration of PSA in the sample.

Results and Discussion

FIG. 7 shows experimental results for the response of total PSAsubtraction assay carried out with wet reagents in a test cartridge. Thetest used 0.7 ul volume and took a total of 10 min, in a buffer matrix.The 0 fM PSA result shows the mean of 4 separate measurements, carriedout on different days with an error of 2 standard deviations. All otherdata points represent single measurements. The x axis represents theconcentration of total PSA in fM. The y axis represents the reactionrate in A/s.

The data shown in FIG. 7 indicates that this assay format gives veryhigh sensitivity, with levels well below 10 fM total PSA easilydetectable from zero control. The data shows a measurable range ofaround 2 orders of magnitude (1.57-155 fM) with a curving responsegiving generally decreasing differentiation between points as the totalPSA level increases.

In the subtraction assay, the rate decreases with increasing total PSAconcentration as the magnet is removing paramagnetic particles complexedto electrochemical label via PSA away from the vicinity of the workingelectrode. As the rate is proportional to the concentration of theelectrochemical label, this decreases as more of it is removed withincreasing total PSA levels. It should be noted that the PSA samplesused in this assay have been calibrated against the World HealthOrganisation (WHO) 1^(st) IRP (96/670) for total PSA.

The sensitivity and measurable range of this assay can be tuned by, forexample, optimisation of reagent composition and concentration, as wellas binding times and measurement times.

The way this assay has been measured there has been no correction forvariations in background (caused by potential variation in reagentvolume, working electrode area etc.). However, it is envisaged that aslight alteration of the measurement could allow an accurate backgroundmeasurement to be made prior to the specific measurement for each samplein each channel. For example, this could be accomplished by starting theelectrochemical measurement earlier at −0.1V (for example after 2 minincubation instead of 4.5 min) and allowing it to reach a steady rateprior to magnet application to the strip. Once the magnet is applied,this rate would change and the change in rate would be proportional tothe PSA concentration, with the initial rate being the backgroundmeasurement.

Here follows more experimental data generated by a specific embodimentof the current invention. The fluorescent detection of signal is made bya commercially available fluorescence plate reader (Perkin Elmer Victor3V) in place of the reader.

Fluorescent Dry Assay of Free PSA

Materials:

Maleimide-PEG2-biotin: Thermo Scientific, Cat 21901 (EZ-linkmaleimide-PEG2-biotin).

Latex particles: Invitrogen, Cat F8827 (FluoSpheres, carboxylatemodified microspheres, 2 μm, yellow-green fluorescent)

Paramagnetic particles: Ademtech, Cat 03223 (200 nm Strep+ magneticparticles)

Antibody 8A6: Hytest, Cat 4P33 MAb 8A6 (Anti-PSA, human)

Antibody 5A6: Hytest, Cat 4P33 MAb 5A6 (Anti-PSA, human)

PBS: Thermo Scientific, Cat 28372 (BupH phosphate buffered saline packs)

BSA: Sigma, Cat A4503-50 G (Albumin, from bovine serum)

Water: Sigma, Cat W4502 (water for molecular biology)

Trehalose: Sigma, Cat T9531-25 G (D-(+)-trehalose dehydrate)

MES: Sigma, Cat M8250-25 G (MES hydrate)

HCl: Sigma, Cat H1758-100 ML (hydrochloric acid, 36.5-38%)

NaOH: Sigma, Cat 72068 (sodium hydroxide solution)

2MEA: Thermo Scientific, Cat 20408 (2-mercaptoethanolaminehydrochloride)

PSA standards (calibrated against WHO 1^(st) IRP (96/670)): PerkinElmer, Cat A073-301 (ProStatus PSA free/total kit, Delfia)

Biotin quantification kit: Thermo Scientific, Cat 28005 (Pierce biotinquantification kit)

Size exclusion columns: GE Healthcare, Cat 17-0851-01 (PD10 columns)

EDTA: Sigma, Cat EDS-100 G (ethylenediamine tetracetic acid, anhydrous)

Tween: Sigma P7949-100 ML (Tween-20)

DMSO: Thermo Scientific, Cat 20684 (dimethylsulfoxide)

Acetic acid: Sigma, Cat 32,009-9 (acetic acid)

Reagent Preparation

Antibody Biotinylation.

Antibody 8A6 Disulphide Bond Reduction

Use undiluted antibody 8A6 stock at a concentration between 2 and 7mg/ml. An appropriate volume of antibody stock is removed to give 1 mgantibody. An appropriate volume of 14.28 mM EDTA in PBS, pH7.2 is addedto 1 mg antibody to give an EDTA concentration of 1 mM.

6 mg of 2MEA is dissolved in 100 ul 1 mM EDTA in PBS, pH7.2. 1 ul ofthis 2MEA solution is added per 10 ul of antibody solution. Thissolution is mixed and incubated in a waterbath at 37 deg for 90 min.

This solution is then passed through a PD10 column (pre-equilibratedwith 1 mM EDTA In PBS, pH7.2) and 500 ul fractions collected. A samplefrom each fraction is taken and measured on UV spectrophotometer, withthe absorbance at 280 nm used to quantify the protein found in eachfraction. The fractions containing significant concentrations of proteinare chosen and combined and remeasured on the UV spectrophotometer. Thismeasurement is used to determine the antibody concentration using anextinction coefficient of the antibody of 1 mg/ml=1.4 absorbance unitsat 280 nm.

Binding of Maleimide-PEG2-Biotin to Antibody

Maleimide-PEG2-biotin is dissolved in 1 mM EDTA in PBS, pH7.2 to give a20 mM solution. An appropriate volume of this is added to the reducedantibody to give a 40 times molar excess of maleimide-PEG2-biotin overreduced antibody. This is then mixed and incubated for 3 hours at roomtemperature.

This is then passed through another PD10 column which has beenpre-equilibrated with 1 mM EDTA in PBS, pH7.2. 500 μl fractions arecollected and measured using the UV spectrophotometer at 280 nm. Thefractions containing significant protein levels are chosen and combined.A sample of this solution is measured again at 280 nm by absorbance, andthe concentration of antibody determined using the extinctioncoefficient of the antibody of 1 mg/ml=1.4 absorbance units at 280 nm.The number of biotins bound per antibody are then determined using thePierce biotin quantification kit, according to the manufacturer'sinstructions.

Latex.

Antibody Adsorption

2 μm latex is washed in MES buffer (50 mM MES, pH6.5) usingcentrifugation at 16100×g for 8 min at 4 degC to pellet the particles.The latex is resuspended at a concentration of 2% solids. Antibody 5A6is prepared at a concentration of 250 μg/ml in MES buffer. 2% latex and250 μg/ml antibody are added together in equal volumes and mixed well.They are incubated for 18 h with mixing on a rotary mixer (30 rpm) atroom temperature. The particles are then washed in an equal volume ofPBS (pH7.2) 3 times (using centrifugation at 16100×g for 8 min, 4 degC)and resuspended in the same at a concentration of 2% solids.

Paramagnetic Particles

Binding of Antibody to Particle

200 nm streptavidin coated paramagnetic particles are washed (using amagnetic separator) in 0.1% tween in PBS, pH7.2 and resuspended in thesame to give a concentration of 0.5% solids. Biotinylated antibody 8A6is diluted in 0.1% tween in PBS, pH7.2 to give 50 μg/ml. An equal volumeof 0.5% paramagnetic particles and 50 μg/ml biotinylated antibody arecombined, mixed and allowed to incubate for 30 min at room temperature,with shaking using a rotary shaker at 30 rpm.

The paramagnetic particles were then washed 4 times (using a magneticseparator) in an equal volume of 0.1% tween in PBS, pH7.2 andresuspended in the same to give a concentration of paramagneticparticles of 1% solids.

Reagent Deposition

Reagents were diluted and combined to give a final deposition solutioncontaining the following:

-   0.02% latex particles functionalised with antibody 5A6, in PBS,    pH7.2-   0.1% paramagnetic particles functionalised with antibody 8A6, in    PBS, pH7.2-   180 mg/ml BSA, in PBS, pH7.2-   1 mg/ml trehalose, in PBS, pH7.2

The cartridge used for the dry assay was that as shown in FIG. 2, exceptwithout any electrodes on the base (23). The base layer (20) andadhesive layer (21) were combined and even pressure applied to sealthese layers together to form a half assembled cartridge.

0.5 ul of deposition solution was deposited within a channel (24) of thehalf assembled cartridge by pipette.

This was dried at 40 degC for 30 min.

The lid (22) was then applied to the half assembled cartridge and evenpressure applied to seal the fully formed cartridge, complete with driedreagents within the channel.

Assay Procedure

2.5 ul of PSA standard was then applied to the cartridge to fill the 4channels and resuspend the dried reagents.

The cartridge was incubated at room temperature for 8 minutes to allowthe binding reaction to occur.

The cartridge was placed on a custom built holder to allow measurementwithin a channel by a Perkin Elmer Victor3 V.

The fluorescent signal in the channel was measured using the inbuiltprogram ‘Fluorescein (485 nm/535 nm, 0.1 s)’. This program usesexcitation at 485 nm and emission at 535 nm with a 0.1 s measurementtime. This signal was recorded as Fln 1.

A magnet was then applied to the cartridge to remove the paramagneticparticles (and any paramagnetic particle-bound components) from thechannel.

The fluorescent signal in the channel was then re-measured using theinbuilt program ‘Fluorescein (485 nm/535 nm, 0.1 s)’. This signal wasrecorded as Fln 2.

The difference in the two signals (Fln 1-Fln 2) was calculated and theresults graphed against free PSA concentration as shown in FIG. 8.

Results and Discussion

The data shown in FIG. 8 indicates that the fluorescent assay formatgives high sensitivity, with free PSA concentration of 58 fM detectablefrom zero control. The data shows a measurable range of around 2 ordersof magnitude (58-5000 fM) with a curving response giving generallydecreasing differentiation between points as the total PSA levelincreases.

In this subtraction assay, the Fln 1 signal is independent of PSAconcentration whereas the Fln 2 signal decreases with increasing freePSA concentration as the magnet is removing paramagnetic particlescomplexed to fluorescent label via PSA from the channel. The differencebetween these signals therefore increases as the PSA concentrationincreases.

It should be noted that the PSA samples used in this assay have beencalibrated against the World Health Organisation (WHO) 1^(st) IRP(96/670) for total PSA.

The sensitivity and measurable range of this assay can be tuned by, forexample, optimisation of reagent composition and concentration, as wellas binding times and excitation and emission wavelengths.

The way this assay and the other assays described herein, has beenmeasured, by using the difference between two measurements corrects thesignal for variations in background (caused by potential variation inreagent volume, working electrode area etc.). This allows a moresensitive and accurate measurement to be made.

Here follows more experimental data generated in support of theadvantages of a specific embodiment of the current invention.

Method Development

A further method has been developed to measure the label glucose oxidase(GOD) (or other enzyme labels) in the described subtraction assay methodwhereby magnetic particle bound label is removed from the detectionarea. The unbound GOD label is therefore measured and used to assignanalyte concentration. The GOD label concentration is measured beforemagnetic separation/removal of the bound GOD label from the detectionzone. The two measurements are then used to calculate the relativechange in label concentration and assign analyte concentration.

During the assay binding time the enzymatic reaction is simultaneouslyoccurring (all events occurring in the detection zone). An example GODdetection cascade is shown in FIG. 11 using ferrocyanide conversion intoferricyanide by peroxidase using hydrogen peroxide. In the examplesdiscussed in this section from here onwards, ABTS is used in the GODdetection cascade instead of ferrocyanide, and is converted to oxidisedABTS by peroxidase using hydrogen peroxide. In this example, therefore,the GOD detection cascade is producing oxidised ABTS during the assaybinding time (e.g. for 4 minutes). Therefore changes of GOD labelconcentration after the magnetic removal of GOD label-magnetic particleanalyte complex from the detection zone are being determined on a largerbackground current. In previous examples we have used rate measurementsto make highly accurate measurements of GOD concentration and henceanalyte concentrations. We have developed an additional electrochemicalmethod that also produces very accurate measurements of GODconcentrations.

A typical GOD titration curve is shown in FIG. 16 whereby the currentvalue plotted is extracted from the 3 second point of a 3 secondtransient, the GOD was allowed to react with the substrate system for 2minutes before a chronoamperometric measurement was performed. A linearresponse is observed. The four channel strip (as shown in FIG. 15) wasused for these measurements.

Therefore after 2 minutes of enzymatic reaction, a defined GODconcentration will produce a certain current. If we allowed theenzymatic reaction to occur for another two minutes the currentsproduced for a defined GOD concentration would be larger. Therefore postmagnetic separation unbound GOD label concentrations are determined onlarge background currents (4 minutes of enzymatic reaction during the 4minutes of assay binding). We have therefore developed the followingtechnique to counteract this scenario.

To prevent large amounts of oxidised ABTS being created by GOD, anelectrochemical potential is applied to the electrodes for the durationof the assay binding (e.g. 4 mins). In this example a −350 mV potentialis applied however the appropriate potential for any enzyme/mediatorsystem could be used. The −350 mV potential applied to the strip during4 minutes allows for 2 fundamental processes to occur. Firstly it allowsa measure of GOD label concentration and secondly it significantlyreduces the amount of oxidised ABTS created during the 4 minute assaybinding time (and hence the current magnitude upon which GOD labelconcentrations are measured).

A GOD titration response is shown in FIG. 17 whereby the current valueplotted is extracted from the 3 second point of a 240 second transient.This response can therefore be used to assign GOD concentrations or inthe context of the immunoassay procedure, a measure of GOD labelconcentration pre magnetic separation (i.e. before the removal ofmagnetic particle bound GOD). This GOD concentration is then used inconjunction with the final unbound GOD label concentration to correctfor variations in GOD label concentrations.

Although the 3 sec time point measurement is used from the 240 sectransient generated, any timepoint from within this transient could beused to assign GOD concentration. After 240 seconds the −350 mVpotential is switched off and the strip returns to open circuitpotential (OCP). In the example we describe here the GOD reaction wasallowed to occur for 2 minutes (OCP, no applied potential), a 3 secondchronoamperometric measurement was then performed. The 3 sec currentvalue from the 3 sec transient was then plotted vs. GOD concentrations(see FIG. 18). A linear, very accurate response is observed. Thismeasurement represents the post magnetic separation measurement ofunbound GOD label concentration. This post magnetic separation GOD labelconcentration and the pre magnetic separation GOD concentration would beused together to determine the analyte concentration.

The GOD titration curves for a normal 2 minute GOD titration and a 2minute titration after the applied −350 mV potential for 4 minutes (240sec) are both shown in FIG. 19. The responses are very similar. Thesmall offset between the responses is driven by differences inelectrochemical background currents and small amount of oxidised ABTSthat will be accumulated during the 240 secs of applied −350 mVpotential. If the 240 secs of −350 mV potential had not been applied,the comparison would be 6 minutes of total GOD turnover vs. 2 minutes ofGOD reaction. In the context of the assay scheme this would be measuringpost magnetic separation GOD concentrations with 4 minutes of currentgenerated during the pre magnetic separation stage of the assay bindingtime.

This measurement methodology was then applied to an immunoassaymeasurement of Total PSA to demonstrate the utility of the measurement.The experiment was combined with reagent changes to show increases inthe dynamic range of the PSA assay.

Here follows more experimental data generated by a specific embodimentof the current invention. The electrochemical detection of signal ismade by a commercially available potentiostat, in place of the reader.

Electrochemical Assay of Total PSA (1)

Materials:

PSA: Hytest, Cat 8P78 (Prostate Specific Antigen)

ABTS: Fluka, Cat 11557

Zeba size exclusion columns: ThermoFisher Scientific, Cat 89882

SPDP: Pierce, Cat 21857

GOD: BBI Enzymes, Cat GO3B3 (Glucose oxidase)

Maleimide-PEG2-biotin: Thermo Scientific, Cat 21901 (EZ-linkmaleimide-PEG2-biotin).

Latex particles: Invitrogen, Cat F8765 (1 um)

Paramagnetic particles: Ademtech, Cat 03223 (200 nm Strep+ paramagneticparticles)

Antibody 1H12: Hytest, Cat 4P33 MAb 1H12 (Anti-PSA, human)

Antibody 5A6: Hytest, Cat 4P33 MAb 5A6 (Anti-PSA, human)

PBS: Thermo Scientific, Cat 28372 (BupH phosphate buffered saline packs)

BSA: Sigma, Cat A4503-50 G (Albumin, from bovine serum)

Water: Sigma, Cat W4502 (water for molecular biology)

MES: Sigma, Cat M8250-25 G (MES hydrate)

Glucose: Sigma, Cat G8270-1 KG (D-(+)-Glucose)

DTT: Pierce, Cat 20290

HCl: Sigma, Cat H1758-100 ML (hydrochloric acid, 36.5-38%)

NaOH: Sigma, Cat 72068 (sodium hydroxide solution)

HRP: BBI Enzymes, Cat HRP4C (Horseradish Peroxidase)

2MEA: Thermo Scientific, Cat 20408 (2-mercaptoethanolaminehydrochloride)

Biotin quantification kit: Thermo Scientific, Cat 28005 (Pierce biotinquantification kit)

Size exclusion columns: GE Healthcare, Cat 17-0851-01 (PD10 columns)

EDTA: Sigma, Cat EDS-100 G (ethylenediamine tetracetic acid, anhydrous)

Tween: Sigma P7949-100 ML (Tween-20)

DMSO: Thermo Scientific, Cat 20684 (dimethylsulfoxide)

Reagent Preparation

Antibody Biotinylation.

Antibody Disulphide Bond Reduction

Antibody 1H12 is reduced using 50 mM 2MEA in 1 mM EDTA in PBS, at 37degC for 90 min. Reduced antibody is passed through PD10 column andcollected in 1 mM EDTA in PBS and fractions found to contain protein (bymeasurement at 280 nm on UV spectrophotometer) pooled. The concentrationof reduced antibody is calculated using the extinction coefficient ofthe antibody of 1 mg/ml=1.4 absorbance units at 280 nm.

Binding of Maleimide-PEG2-Biotin to Antibody

Maleimide-PEG2-biotin is added to the reduced antibody in molar excessto allow efficient binding to occur and incubated for 3 hours at roomtemperature. This is then passed through another PD10 column which hasbeen pre-equilibrated with 1 mM EDTA in PBS, pH7.2. 500 ul fractions arecollected and measured using the UV spectrophotometer at 280 nm. Thefractions containing significant protein levels are chosen and combined.A sample of this solution is measured again at 280 nm by absorbance, andthe concentration of antibody determined using the extinctioncoefficient of the antibody of 1 mg/ml=1.4 absorbance units at 280 nm.The number of biotins bound per antibody are then determined using thePierce biotin quantification kit, according to the manufacturer'sinstructions.

Latex.

Binding of GOD/1H12 to Latex Via SPDP

Latex particles are bound to SPDP in 1 mM EDTA in PBS buffer at aconcentration of 1% solids and 1 mM SPDP. This is incubated with gentleshaking for 90 min at room temperature in the dark. GOD/5A6 conjugate isbound to SPDP using a molar excess of SPDP:GOD/1H12 and incubated for 90min at room temperature in the dark. After 90 min a proportion of theGOD/5A6-SPDP is reduced by the addition of DTT at pH4.5 with a further30 min incubation at room temperature in the dark. After the specifiedbinding time, the latex particles are washed into 1 mM EDTA in PBSbuffer at a concentration of 1% solids (using centrifugation andsonication for pelleting and resuspension respectively). TheGOD/5A6-SPDP reactions are passed through a Zeba desalting column andcollected in 1 mM EDTA in PBS. The latex-SPDP and GOD/5A6-SPDP are thencombined to give an excess of GOD/5A6-SPDP to latex-SPDP binding sites.This binding reaction is incubated for 19 h at room temperature withgentle shaking in the dark. This binding reaction is then washed into0.1% tween in PBS and resuspended at 1% solids and stored at +4 degC inthe dark until use.

Paramagnetic Particles

Binding of Antibody to Particle

200 nm streptavidin coated paramagnetic particles are washed (using amagnetic separator) in 0.1% tween in PBS, pH7.2 and added tobiotinylated antibody 1H12 to allow binding of the biotin of thebiotinylated antibody to the streptavidin-coated paramagnetic particle,with a 1 h10 min incubation at room temperature with gentle shaking. Theparamagnetic particles are then washed 4 times (using a magneticseparator) in an equal volume of PBS, pH7.2 and resuspended in the sameto give a concentration of paramagnetic particles of 1% solids which arestored at +4 degC until use.

Assay Procedure

Assay reagents were added to an eppendorf tube in the following volumesand concentrations:

-   1% paramagnetic particles (with bound b1H12): 1 ul-   1% Latex (with bGOD and b5A6 bound): 1 ul-   PSA (10× final concentration in 300 mg/ml BSA in PBS): 1 ul

All reagents added to eppendorf separately and kept separate in tube.All reagents finally combined by mixing upon addition of the:

-   Reaction buffer: 7 ul-   (Reaction buffer comprises 500 mM MES, pH 6.7, 250 mM glucose, 15 mM    ABTS, 4 mg/ml HRP, 0.14% tween)

These reagents are mixed thoroughly then 4 ul is added to a cartridge,which is connected up to a potentiostat (Autolab PGSTAT12). Theimmunoassay reaction contains all the reagents for the binding reaction(anti-PSA antibody magnetic particles, PSA analyte, anti-PSAantibody-GOD label) and all the reagents for the enzymatic reaction(glucose, HRP, ABTS). The cartridge is made up of 4 channels, with 3channels each spanned by 2 screen-printed carbon electrodes (one workingelectrode and one counter/reference electrode) and 1 channel beingspanned by 2 screen printed silver/silver chloride electrodes as shownin FIGS. 1 and 15. Measurements were carried out in the 3 channels withscreen printed carbon electrodes in this experiment, as follows:

Experimental Methodology

As soon as the reagents are mixed the binding and enzymatic reactionbegin. This all occurs in the detection zone. After 30 seconds anegative 350 mV potential is applied to the strip for 240 seconds (asshown in the transient). The 3 second current value from the 240 sectransient is recorded (other points from the transient could be used).This value represents a measure of the anti-PSA antibody-GOD labelconcentration at the detection zone (working electrode) of the strip andis called the pre magnetic separation current/measurement. After 210seconds the magnetic is applied to the strip. The anti-PSA antibodymagnetic particles-PSA-anti-PSA antibody-GOD label complexes are removedfrom the detection zone leaving behind unbound anti-PSA antibody-GODlabel. The −350 mV potential is switched off after 240 seconds (afterthe magnetic separation step) returning the detection zone/strip to OCP.The strip remains at OCP for 2 minutes before a chronoamperometricmeasurement is performed. The 3 second current value from a 3 secondtransient was then recorded; this is called the post magnetic separationcurrent/measurement. This procedure was repeated for all the PSAconcentrations assayed.

Results and Discussion

The non corrected post magnetic separation measurement of PSAconcentration is shown in FIG. 20. A systematic decrease in current isobserved as the concentration of PSA is increased. The precision isfairly good however at low PSA concentrations although the means aresystematic with PSA concentration the associated error is bad.

The variation in the PSA assay results are driven by variations in thepre magnetic separation current/measurement. This is shown in thefollowing analysis. The difference between post magnetic separationcurrent and the pre magnetic separation current for all concentrationsand reps are shown in FIG. 21. This data is graphically represented inFIG. 22

The PSA assay performance has improved compared to the uncorrected postmagnetic separation current with increased sensitivity and overallprecision. The PSA assay improves further when the proportionality ofchange between post magnetic separation current and pre magneticseparation current are taken into consideration. The ratio of the postmagnetic separation current to the pre magnetic separation current isshown in FIG. 23, the graphically representation of this data is shownin FIG. 24. The PSA performance improves further with regards tosensitivity and precision. Although the majority of the pre magneticseparation current discussion is based around the influence of anti-PSAantibody-GOD label, we fully recognise the pre magnetic response/currentused for normalisation is the result of the interplay of all inputtingfactors (anti-PSA antibody-GOD label, working electrode size, mediatorconcentrations, cell height, HCT, etc) and therefore used to correct formany sources of assay variation.

The strip format (4 channel, 20 channel etc) allows multiplemeasurements of the same analyte to be made or multiple measurements ofthe same analyte made. Even a crude correction of subtracting the meanpre magnetic separation current (three measurements within a strip) fromthe mean post magnetic separation current (three measurements within astrip) improves assay performance (see FIG. 25). In this contextalthough the assay has been used to measure the analyte 3 times (withinone strip) a mean response would be used to determine the analyte/PSAconcentration of the sample. As a result very accurate measurements onanalyte dose will be observed, due to precision improvements within ameasurement (this is discussed elsewhere in the document). However evenin a mean crude correction methodology a measurement of the pre magneticseparation current is very beneficial.

Here follows more experimental data generated by a specific embodimentof the current invention. The electrochemical detection of signal ismade by a commercially available potentiostat, in place of the reader.

Electrochemical Assay of Total PSA (III)

Materials:

EDC: Sigma, Cat 03449

Sulfo-NHS: ThermoFisher Scientific, Cat 24510

Latex particles: Invitrogen, Cat F8827 (2 um, COOH surface)

Antibody 1H12: Hytest, Cat 4P33 MAb 1H12 (Anti-PSA, human)

Antibody 5A6: Hytest, Cat 4P33 MAb 5A6 (Anti-PSA, human)

GOD: BBI Enzymes, Cat GO3B3 (Glucose oxidase)

Paramagnetic particles: Chemicell 1, 500 nm SiMag-Carbonyl

PBS: Thermo Scientific, Cat 28372 (BupH phosphate buffered saline packs)

BSA: Sigma, Cat A4503-50 G (Albumin, from bovine serum)

Water: Sigma, Cat W4502 (water for molecular biology)

Sodium acetate: Sigma, Cat 58750

MES: Sigma, Cat M8250-25 G (MES hydrate)

Tris: Sigma, Cat 93362 (Trizma base)

Ferrocyanide: Sigma, Cat P3289-100 G (potassium ferrocyanide)

Glucose: Sigma, Cat G8270-1 KG (D-(+)-Glucose)

HCl: Sigma, Cat H1758-100 ML (hydrochloric acid, 36.5-38%)

NaOH: Sigma, Cat 72068 (sodium hydroxide solution)

HRP: BBI Enzymes, Cat HRP4C (Horseradish Peroxidase)

PSA: Hytest, Cat 8P78 (Prostate Specific Antigen)

EDTA Sigma, Cat EDS-100 G (ethylenediamine tetracetic acid, anhydrous)

Tween: Sigma P7949-100 ML (Tween-20)

DMSO: Thermo Scientific, Cat 20684 (dimethylsulfoxide)

Acetic acid: Sigma, Cat 32,009-9 (acetic acid)

Trehalose: Sigma, Cat T9531 (D-(+)-trehalose dihydrate)

Reagent Preparation

Latex Binding to GOD and 5A6 Using EDC

Incubate 1% latex particles with 20 mg/ml EDC and 20 mg/ml sulfo-NHS,all in 50 mM MES buffer, pH6.0 for 20 min at room temperature in thedark. Wash latex particles in 50 mM sodium acetate, pH4.6 and resuspendin same at 1% solids.

Add latex-EDC to GOD and 5A6 antibody to give final concentrations of0.5% latex, 1.5 mg/ml GOD and 0.5 mg/ml 5A6 all in 50 mM sodium acetate,pH4.6. Incubate for 3 h10 min at room temperature with gentle shaking.

Wash latex particles in 50 mM tris buffer, pH7.1 and incubate at 4 degCovernight. Wash latex particles in 0.1% tween in PBS and resuspend insame at 0.5% solids. Store at 4 degC in dark until use.

Paramagnetic Particle Binding to Antibody 1H12 Using EDC

Incubate 1% paramagnetic particles with 20 mg/ml EDC and 20 mg/mlsulfo-NHS, all in 50 mM MES buffer, pH6.0 for 20 min at room temperaturewith gentle shaking. Wash with PBS and resuspend in same at 1% solids.

Incubate 0.5% paramagnetic particles-EDC with 0.5 mg/ml 1H12, all inPBS, pH7.2, for 2 h15 min at room temperature.

Wash in 0.1% tween in 50 mM tris buffer at pH7.1 and incubate at 4 degCovernight. Wash in 0.1% tween in PBS and resuspend in same to give 0.5%solids. Store at 4 degC until use.

Assay Procedure

Assay reagents were added to an eppendorf tube in the following volumesand concentrations:

-   1% paramagnetic particles (with bound 1H12): 1 ul-   0.5% Latex (with GOD and 5A6 bound): 1 ul-   PSA (5× final concentration in 60 mg/ml BSA in PBS): 2 ul-   1 M glucose: 1 ul

All reagents added to eppendorf separately and kept separate in tube.All reagents finally combined by mixing upon addition of the:

-   Reaction buffer: 5 ul-   (Reaction buffer comprises 1 M MES, pH 6.0, 2 mg/ml trehalose, 120    mg/ml BSA, 4 mg/ml HRP, 200 mM ferrocyanide)

These reagents are mixed thoroughly then 0.8 ul is added to a cartridge,which is connected up to a potentiostat (Autolab PGSTAT12). Thecartridge is made up of a single channel spanned by 2 screen-printedcarbon electrodes (one working electrode and one counter/referenceelectrode) as shown in FIGS. 3 and 4. Measurements were carried out asfollows:

Experimental Methodology

A pulsed electrochemical methodology was used to measure theanti-PSA-GOD label before and after the magnetic separation step. Achronoamperometric measurement was made every minute for a total of 8minutes. Specifically a 0.1 sec chronoamperometric measurement at −350mV was performed; the current value at the 0.1 seconds was recorded fromthe 0.1 second transient. After the application of the sample to thestrip the binding and enzymatic reactions are simultaneously occurring.The pulsed electrochemical measurements are then performed every 1minute and the data recorded. After 4 minutes the magnetic field isapplied to the strip and the anti-PSA magnetic particles are removedfrom the working electrode. The removed anti-PSA magnetic particles willbe a mixed population of anti-PSA magnetic with bound anti-PSA-GOD labelvia PSA and anti-PSA magnetic particles (with no label bound). Thepulsed electrochemical measurements for 5, 6, 7 and 8 minutes constitutethe measurement of the remaining unbound anti-PSA-GOD label. The 2, 3and 4 minute currents values were used to calculate a slope by plottingTime (mins) vs. Current (amps) and fitting a linear regression. Thisslope constitutes the pre magnetic separation measurement.

The same methodology was applied to the 5, 6, 7 and 8 minute data. Theresultant slope constitutes the post magnetic separation measurement asis a measure of the unbound anti-PSA-GOD label.

Results and Discussion

The ratio of the pre magnetic separation slope divided by the postmagnetic measurement was then plotted against Total PSA concentration(pM) as shown in FIG. 26. A systematic linear response is observed, withhigh sensitivity.

What is claimed is:
 1. A method of conducting an assay on a sample, the method comprising the steps of: introducing a fluid sample into a microfluidic cartridge such that any analyte present in the sample is capable of being bound by an analyte binding agent and a label present in the cartridge, the cartridge comprising: a sample port for introducing said sample into the cartridge, a substrate comprising one or more microfluidic channels disposed therein, a binding agent within said channel(s) for binding any of said analyte within the sample, and a label for use in detecting an amount of the analyte present in the sample; and a detection area within or wholly comprising an area in which analyte, analyte binding agent, and label binding occurs to produce analyte binding agent/analyte/label complexes , detecting a total level of the label that is complexed or uncomplexed to the analyte present in the detection area in order to obtain a first reference/control value; removing any analyte binding agent/analyte/label complexes from the detection area; detecting a level of any uncomplexed label and any unreacted label that remains in the detection area after removal of said analyte binding agent/analyte/label complexes; and determining said analyte concentration by subtracting the level of uncomplexed/unreacted label from the first reference/control value.
 2. The method according to claim 1, wherein introduction of the sample to the cartridge causes at least a portion of the binding agent and the label to be brought into solution or suspension within the detection area.
 3. The method according to claim 2, wherein the detection is carried out electrochemically.
 4. The method according to claim 3, wherein an oxidising potential or a reduction potential is used to measure a level of the label.
 5. The method according to claim 4, wherein the oxidising potential or the reduction potential is applied to reduce any background effects, such as to eliminate or minimise effect of electrochemical interferents which may be present in the sample, wherein the effect of the electrochemical interferents may include generating a background signal or minimising a current generated by an enzyme during the binding incubation phase.
 6. The method according to claim 3, wherein glucose oxidase is employed as the label.
 7. The method according to claim 6, further comprising the step of: using a peroxidise and a mediator wherein the mediator is capable of conversion between an oxidised form and a reduced form and is capable of electrochemical detection.
 8. The method according to claim 1, wherein the binding agent is coupled or otherwise bound to a particle which is capable of being complexed with the analyte present in the sample and which can be removed, when complexed or uncomplexed with the analyte, from the detection area, by suitable means.
 9. The method according to claim 8, wherein the particle is a paramagnetic particle and the suitable means is a magnet or an electromagnet.
 10. The method according to claim 1, wherein the sample is a sample of whole blood.
 11. The method according to claim 1, wherein detection is carried out directly on the sample, without any separation, washing step, or dilution of the sample by a further fluid being carried out.
 12. The method according to claim 1, wherein a decrease in a signal before and after removal of the bound analyte/label from the detection area is detected, and wherein the signal is proportional to the amount of the analyte present in the sample.
 13. The method according to claim 7, wherein the peroxidase is HRP.
 14. The method according to claim 7, wherein the mediator is ferrocyanide or ABTS. 